The Control of Microbial Growth

Chapter 7
 The Terminology of Microbial Control
• Control of microbial growth can prevent
infections and food spoilage.

• Sterilization is the process of destroying all

microbial life on an object.

• Commercial sterilization is a heat treatment of

some canned foods to destroy C. botulinum
endospores and spoilage microbes.
The Terminology of Microbial Control
- Continue
• Disinfection is the process of reducing or inhibiting
microbial growth on a nonliving surface.

• Antisepsis is the process of reducing or inhibiting

microorganisms on living tissue.

• The suffix -cide means to kill; the suffix -stat means

to inhibit.

• Bacterial contamination is called sepsis or when

referring to a disease state, it refers to gram-negative
bacteria in the bloodstream.
 The Rate of Microbial Death
• Bacterial populations subjected to heat or
antimicrobial chemicals usually die at a
constant rate.
• Such a death curve, when plotted
logarithmically, shows this constant death
rate as a straight line.
• Time to kill a microbial population is
proportional to the number of microbes
present.
 The Rate of Microbial Death _
Continue
• Vegetative forms of microbes are usually more
susceptible to physical and chemical controls
as compared to endospores.
• Organic matter may interfere with heat
treatments and chemical control agents.
• Longer exposure to lower temperature can
produce the same (equivalent) lethal effect as
shorter time at higher temperature.
Actions of Microbial Control Agents
• Alteration of Membrane Permeability
– The susceptibility of the plasma membrane is due
to its lipid and protein components.
– Certain chemical control agents damage the
plasma membrane by altering its permeability.
• Damage to Proteins and Nucleic Acids
– Some microbial control agents damage cellular
proteins by breaking hydrogen and covalent bonds.
– Other agents interfere with DNA and RNA
replication and protein synthesis
Physical Methods of Microbial Control

• High Temperature (Thermobacteriology)

• Filtration
• Low Temperature
• Desiccation
• Osmotic Pressure.
• Radiation
 High Temperature
• High Temperature is used to kill or destroy
microorganisms.
• Moist heat kills microbes by denaturing
enzymes.
• Thermal death point (TDP) is the lowest
temperature at which all bacteria in a liquid
culture will be killed in 10 minutes.
• Different methods that produce the same
reduction in microbial growth or population
are called equivalent treatments.
 High Temperature - Continue

• Thermal death time (TDT) is the length of

time required to kill all bacteria in a liquid
culture at a given temperature.
• Decimal reduction time (DRT) or D value is
the time a certain temperature to reduce a
microbial population by 90% (or 10 fold or by
one log).
 Survivor Curve

8
Bacterial Population (log cfu/g or ml)

5
D
4

3
0 5 10 15 20 25
Time (min)
 High Temperature - Continue
• Boiling (100°C) kills many vegetative cells
and viruses within 10 minutes.
• Autoclaving (121°C for 15 min; steam under
pressure: 15 pound per square inch) is the
most effective method of moist heat
sterilization. The steam must directly contact
the material to be sterilized. Autoclave
Autoclave
 Heat
• In Milk pasteurization:
– LTLT: Low Temperature Long Time (63C, 30 min)
– HTST: High Temperature Short Time (72C, 15 sec)
– UHT: Ultra High Temperature (140°C for 3 sec.) is used
to “sterilize” dairy products.
• Methods of dry heat sterilization:
– Direct flaming, incineration, and hot-air sterilization.
– Requires higher temperatures (160-170C or 320-338F for
periods of 2 to 4 hours) as compared to autoclaving (121-
132C or 250-270F for 15 to 40  minutes).
– Dry heat kills by oxidation.
 Filtration
• Filtration is the passage of a liquid or gas
through a filter with pores small enough to
retain microbes (0.45 µ to 0.2 µ for bacteria).
• Microbes can be removed from air by high-
efficiency particulate air filters.
• Membrane filters composed of nitrocellulose
or cellulose acetate are commonly used to
filter out bacteria, viruses, and even large
proteins.
Filtration
 Low Temperature
• The effectiveness of low temperatures depends on the
particular microorganism and the intensity of the
application.
• Most microorganisms (except for the psychrophiles)
do not reproduce at ordinary refrigerator
temperatures (0°-7°C).
• Many microbes survive (but do not grow) and some
die at the subzero temperatures used to store foods (-
18C or 0F and below).
• Microorganisms are well preserved in liquid nitrogen
(-196C) - a common method of preserving bacterial
strains in research laboratories.
 Desiccation Osmotic Pressure
• Microorganisms in high
• In the absence of water, concentrations of salts
microorganisms cannot and sugars undergo
grow but can remain plasmolysis.
viable. • Molds and yeasts are
• Viruses & endospores more capable than
can resist desiccation. bacteria of growing in
• Salmonella survives dry environments with low
conditions moisture or high
osmotic pressure.
 Radiation
• The effects of radiation depend on its wavelength, intensity,
and duration.
• Ionizing radiation:
– gamma rays, X rays, and high-energy electron beams.
– high-energy and a high degree of penetration and exerts its effect
primarily by ionizing water and forming highly reactive hydroxyl
radicals.
• Ultraviolet (UV) radiation:
– a form of nonionizing radiation, has a low degree of penetration
– causes cell damage by making thymine dimers in DNA that interfere
with DNA replication
– the most effective germicidal wavelength is 260 nm.
– Used to sterilize air, surfaces and may be water
• Microwaves can kill microbes indirectly as materials get hot.
Bacterial spores are resistant.
 Chemical Methods of Microbial
Control
• Chemical agents are used on living tissue (as antiseptics) and
on inanimate objects (as disinfectants).
• Few chemical agents achieve sterility:
– 50% to 70% ethanol
– Phenolic compounds - Lysol.
– Tincture of iodine (2% I in 70% alcohol)
– Ethylene oxide gas
– Chlorine
• Principles of Effective Disinfection
– Careful attention should be paid to the properties and concentration of
the disinfectant to be used.
– The presence of organic matter, degree of contact with
microorganisms, and temperature should also be considered.
– Chlorine bleach effectiveness is reduced by organic matters
 Evaluating a Disinfectant
• Use-dilution test: bacterial (Salmonella choleraesuis,
Staphylococcus aureus, and Pseudomonas
aeruginosa) survival in the manufacturer's
recommended dilution of a disinfectant is determined.
– Viruses, endospore forming bacteria, mycobacteria, and
fungi can also be used in the use-dilution test.
• Minimum inhibitory concentration (MIC)
• Disk-diffusion method: a disk of filter paper is
soaked with a chemical and placed on an inoculated
agar plate; a clear zone of inhibition indicates
effectiveness.
Assessing the antimicrobial activity of a compound using the minimum inhibitory concentration
method A compound to be tested is serially diluted into growth medium, inoculated with a culture and
then incubated. The minimum inhibitory concentration is indicated in the lowest
dilution of the compound which prevents growth as indicated by the arrow.
Disk-Diffusion Method

Bacillus cereus
inoculated soaked;
alcohol no effect
 Types of Disinfectants
• Phenol and Phenolics
– Phenolics exert their action by injuring plasma membranes.
• Bisphenols
– Bisphenols such as triclosan (over the counter) and
hexachlorophene (prescription) are widely used in
household products.
• Biguanides
– Chlorhexidine damages plasma membranes of vegetative
cells.
 Halogens
• Some halogens (iodine and chlorine) are used alone or as
components of inorganic or organic solutions:
– Iodine may combine with amino acids to inactivate enzymes & cellular
proteins.
– Iodine is available as a tincture (in solution with alcohol) or as an
iodophor (combined with an organic molecule).
• The germicidal action of chlorine is based on the formation of
hypochlorous acid (HOCl) when chlorine is added to water.
• Chlorine is used as a disinfectant in gaseous form (Cl2) or in
the form of a compound, such as:
– calcium hypochlorite; Ca(OCl)2,
– sodium hypochlorite; NaOCl,
– chloramines; monochloramine (NH2Cl), dichloramine (NHCl2) and
trichloramine (NCl3).
 Alcohols
• Alcohols exert their action by denaturing
proteins and dissolving lipids.
• In tinctures, they enhance the effectiveness of
other antimicrobial chemicals.
• Aqueous ethanol (60-90%) and isopropanol
are used as disinfectants. Aqueous ethanol
70% is considered highly disinfectant.
 Heavy Metals and Their Compounds

• Silver, mercury, copper, and zinc are used as

germicidals.
• They exert their antimicrobial action through
oligodynamic (active in very small quantities)
action. When heavy metal ions combine with
sulfhydryl (-SH) groups, proteins are
denatured.
 Surface-Active Agents
• 1. Surface-active agents decrease the tension between
molecules that lie on the surface of a liquid; soaps
and detergents are examples.
• 2. Soaps have limited germicidal action but assist in
the removal of microorganisms through scrubbing.
• 3. Acid-anionic detergents (a long carbon chain to
which a sulfo group (-SO3) is attached, forming the
negatively charged, anionic, part) are used to clean
dairy equipment.
Quaternary Ammonium Compounds
(Quats)

• 1. Quats are cationic detergents attached to

NH4+.
• 2. Disrupt the plasma membranes: cytoplasmic
constituents leak out of the cell.
• 3. Quats are most effective against gram-
positive bacteria.
 Chemical Food Preservatives

• 1. SO2, sorbic acid, benzoic acid, and

propionic acid inhibit fungal metabolism
and are used as food preservatives.
• 2. Nitrate and mainly nitrite salts prevent
germination of Clostridium botulinum
endospores in meats.
 Antibiotics Aldehydes
• Nisin and natamycin are • Aldehydes such as
antibiotics used to formaldehyde and
preserve foods, glutaraldehyde exert
especially cheese. their antimicrobial
• Natamycin is approved effect by inactivating
by the FDA for use on proteins.
the surface of cuts and • They are among the
slices of cheese most effective chemical
disinfectants.
 Gaseous Peroxygens (Oxidizing
Chemosterilizers Agents)
• 1. Ethylene oxide is the • 1. Ozone, peroxide, and
gas most frequently peracetic acid (aka
used for sterilization. Peroxyacetic acid
• 2. It penetrates most C2H4O3) are used as
materials and kills all antimicrobial agents.
microorganisms by • 2. They exert their
protein denaturation. effect by oxidizing
molecules inside cells.
 Microbial Characteristics and
Microbial Control
• 1. Gram-negative bacteria are generally more resistant
than gram-positive bacteria to disinfectants and antiseptics.
• 2. Mycobacteria, endospores, and protozoan cysts and
oocysts are very resistant to disinfectants and antiseptics.
• 3. Nonenveloped viruses are generally more resistant than
enveloped viruses to disinfectants and antiseptics.
• 4. Prions (proteinaceous infectious particles)
– Incorrectly folded proteins
– Infectious protein agents (BSE or mad cow disease).
– Prions are abnormally-structured forms of a host protein, which are
able to convert normal molecules of the protein into the abnormal
structure.
– Prions are resistant to disinfection and autoclaving.
Prions